Method for prophylaxis of influenza using vaccine for intranasal administration

ABSTRACT

The present invention provides a vaccine composition for transnasal mucous membrane administration, which contains an influenza virus antigen, polyriboinosinic polyribocytidylic acid (poly (I:C)) or a derivative thereof and a carboxyvinyl polymer. The present invention also provides a prophylactic method of influenza, including a step of administering the vaccine composition at least once to the nasal mucosa of a subject in need thereof.

TECHNICAL FIELD

The present invention relates to a vaccine for the prophylaxis of influenza, and a method for the prophylaxis of influenza.

BACKGROUND ART

Influenza is a viral infectious disease that repeatedly becomes epidemic with different antigen every year. The vaccines approved in Japan for the prophylaxis of influenza are those for subcutaneous administration. Since the vaccination induces an IgG antibody having a neutralizing activity in the serum, it is highly effective for preventing progression of the condition into a more severe one such as pneumonia and the like. In the upper airway mucosa, which is the infection site, however, IgA is the main prophylactic component. Since IgA is not induced by subcutaneous administration, the infection-preventive effect is not sufficient. Therefore, the development of an infection-preventive vaccine has been desired for a long time.

The present inventors have developed an influenza vaccine for mucosal administration comprising a double-stranded RNA and an influenza virus antigen, which is superior in the infection-preventive effect (WO2005/014038).

DISCLOSURE OF THE INVENTION

It is therefore an object of the present invention to provide a novel vaccine which is useful for the prophylaxis of influenza, and a method for the prophylaxis of influenza.

To achieve the above-mentioned object, the present inventors have tried to examine the effects of vaccines by adding various components, and completed the present invention.

Accordingly, the present invention provides the following.

-   [1] A vaccine composition for nasal mucosal administration,     comprising an influenza virus antigen, polyriboinosinic     polyribocytidylic acid (poly (I:C)) or a derivative thereof and a     carboxyvinyl polymer. -   [2] The composition of [1], wherein the aforementioned antigen is a     subunit antigen or an inactivated antigen. -   [3] The composition of [2], wherein the aforementioned subunit     antigen comprises at least one subunit antigen selected from the     group consisting of HA, NA, M1, M2, NP, PB1, PB2, PA and NS2 of     influenza virus. -   [4] The composition of [2], wherein the aforementioned subunit     antigen comprises at least one subunit antigen selected from the     group consisting of HA and NA. -   [5] The composition of [1], wherein the aforementioned derivative is     poly (I:C₁₂U). -   [6] A method of preventing influenza, comprising a step of     administering a vaccine composition comprising an effective amount     of an influenza virus antigen and poly (I:C) or a derivative     thereof, and a carboxyvinyl polymer at least once to the nasal     mucosa of a subject in need thereof. -   [7] The method of [6], wherein the aforementioned antigen is a     subunit antigen or an inactivated antigen. -   [8] The method of [7], wherein the aforementioned subunit antigen     comprises at least one subunit antigen selected from the group     consisting of HA, NA, M1, M2, NP, PB1, PB2, PA and NS2 of influenza     virus. -   [9] The method of [7], wherein the aforementioned subunit antigen     comprises at least one subunit antigen selected from the group     consisting of HA and NA. -   [10] The method of [6], wherein the aforementioned derivative is     poly (I:C₁₂U). -   [11] The method of [6], wherein the aforementioned vaccine     composition is administered at least twice. -   [12] The method of [11], wherein the aforementioned vaccine     composition is administered at an interval of at least 1 week.

The characteristics and the advantages of the present invention will be apparent from the detailed description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an immune response in a nasal administration test of an influenza vaccine comprising Ampligen and a CVP base in combination.

FIG. 2 is a flow chart of mouse immunity test in Example 2.

FIG. 3 is a summary of the vaccine administration test in Example 2.

FIG. 4 is a flow chart of monkey immunity test in Example 3.

FIG. 5 shows the results of Serum IgG ELISA in Example 3.

FIG. 6 shows the results of nasal swab IgA ELISA in Example 3.

FIG. 7 shows a summary of the vaccine administration test in Example 3.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, the influenza virus includes any subtype known at present, and subtypes which will be isolated and identified in the future. Since no epidemic has been heretofore observed in human and human infection needs to be effectively prevented hereafter, the influenza virus is preferably a subtype consisting of a combination of a type selected from H1-H16 excluding H1 and H3 (i.e., H2 and H4-16) and a type selected from N1-N9. These subtypes are also called new type of influenza virus. The aforementioned subtype is more preferably a subtype consisting of a combination of a type selected from H5, H7 and H9 and a type selected from N1-N9. The influenza virus may be one type of strain belonging to the same subtype, or two or more types of strains belonging to the same subtype, or two or more types of strains belonging to different subtypes.

The influenza virus antigen contained in the vaccine composition of the present invention is largely divided into an inactivated antigen and a subunit antigen.

The term “inactivated antigen” as used herein refers to an antigen deprived of infectivity, used as a vaccine antigen; such antigens include, but are not limited to, complete virus particle virions, incomplete virus particles, virion-constituting particles, virus non-structural proteins, antigens that protect against infections, neutralizing reaction epitopes and the like. The term “inactivated antigen” as used herein refers to an antigen deprived of infectivity, but retaining immunogenicity; when such an antigen is used as a vaccine, it is called an “inactivated vaccine.” Examples of the inactivation methods of antigens include, but are not limited to, physical (e.g., X-ray irradiation, heat, ultrasound), chemical (formalin, mercury, alcohol, chlorine) or other procedures. Subunit antigen per se also falls within the definition of inactivated antigen because they have usually lost infectivity. Alternatively, a killed virus may be used.

The term “subunit antigen” as used herein refers to a component derived from an influenza virus. The subunit antigen includes hemagglutinin (HA), neuraminidase (NA), matrices (M1, M2), non-structures (NS), polymerases (PB1, PB2: basic polymerases 1 and 2, acidic polymerase (PA)), nuclear proteins (NP) and the like, with preference given to HA or NA, which is exposed to the surface of the virus particle. Currently known types of HA are HA1 to HA16, and known types of NA are NA1 to NA9. The subunit antigen may be purified from a pathogen such as a naturally occurring virus, or may be prepared by a synthetic or recombinant technology. Such methods are well known and in common use in the art, and can be performed using commercially available equipment, reagents, vectors and the like.

The amount of the influenza virus antigen to be contained in the vaccine composition of the present invention is not particularly limited as long as it is sufficient to produce secretary IgA, and can be appropriately determined in consideration of the ratio to the below-mentioned poly (I:C) or a derivative thereof. When HA is used as an antigen, for example, its concentration is preferably 10-500 μg HA/mL (based on HA), more preferably 30-400 μg HA/mL (based on HA). The aforementioned concentration is obtained by measuring the concentration of HA protein.

Poly(I:C) as contained in the vaccine composition of the present invention is a double-stranded RNA (dsRNA) comprising polyinosinic acid (pI) and polycitidic acid (pC).

A derivative of poly (I:C) refers to a mismatched dsRNA obtained by modifying the specific configuration of poly (I:C) through the introduction of unpaired bases thereinto, and includes poly (I:C_(x)U), poly (I_(x)U:C) (where x is on average a number from 3 to 40) and the like. Preferably, a derivative of poly (l:C) is poly (I:C₁₂U) or poly (C:I₁₂U), which is commercially available under the trade name Ampligen™.

Poly (l:C) or a derivative thereof is supplied in a size sufficient to produce secretory IgA. Examples of such sizes include 100 bp or more, with preference given to sizes of 300 bp or more, which sizes, however, are not to be construed as limiting. Examples of the upper limit of size include, but are not limited to, 10⁸ bp.

Poly (I:C) or a derivative thereof is present at a concentration sufficient to produce secretory IgA. Such a concentration of poly (I:C) or a derivative thereof is, for example, 0.1 to 10 mg/mL, more preferably 0.5 to 2 mg/mL, and still more preferably about 1 mg/mL (e.g., 0.8 to 1.2 mg/mL).

The weight ratio of the influenza virus antigen and poly (I:C) or a derivative thereof to be contained in the vaccine composition of the present invention is recommended to be 1:1-1:50.

A carboxyvinyl polymer (CVP) contained in the vaccine composition of the present invention is a hydrophilic polymer which is produced by polymerization of acrylic acid as the main monomer component and includes conventional ones such as Carbopol™ commercially available from Lubrizol Advanced Materials, Inc. US and the like. The concentration of CVP used in the present invention is generally in the range of 0.1-2.0% by weight.

The vaccine composition of the present invention may contain a water-soluble basic substance for the purpose of thickening CVP. The water-soluble basic substance includes, for example, inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonia, etc.), and organic bases such as alkylamines (e.g., methylamine, ethylamine, propylamine, etc.), dialkylamines (e.g., dimethylamine, diethylamine, dipropylamine, etc.), trialkylamines (e.g., trimethylamine, triethylamine, tripropylamine, etc.), alkanolamines (e.g., methanolamine, ethanolamine, propanolamine, etc.), dialkanolamines (e.g., dimethanolamine, diethanolamine, dipropanolamine, etc.), trialkanolamines (e.g., trimethanolamine, triethanolamine, tripropanolamine, etc.), amino acids (e.g., arginine, lysine, ornithine, etc.) and the like. These water-soluble bases are used in an amount which is necessary for neutralization to adjust the pH value of CVP aqueous solution to a desired pH.

The pH value of the vaccine composition of the present invention is adjusted to the desired pH with a water-soluble basic substance or other pH adjustors taking into consideration the stability or absorption of an influenza virus antigen. Preferable pH range is 6.0-8.0.

Adjustment of the viscosity can be performed depending on the vaccine dosage form. For example, CVP corresponding to 0.1-2.0% by weight is thickened with a water-soluble basic substance and the viscosity is adjusted by applying an outside shearing force, or the viscosity is adjusted with a viscosity modulating agent and an outside shearing force, whereby a base managed to suit the spray angle and spray density from a sprayer is prepared. Thereafter, an influenza virus antigen and poly (I:C) are admixed.

The vaccine composition of the present invention may contain a suitable active medicament, diluent, bactericide, preservative, surfactant, stabilizer and the like which can be used together with the vaccine.

The present invention relates to a method of preventing influenza, comprising a step of administering a vaccine composition comprising an effective amount of an influenza virus antigen and poly (I:C) or a derivative thereof, and a carboxyvinyl polymer at least once to the nasal mucosa of a subject in need thereof.

The subject of administration of the vaccine composition of the present invention includes, but is not limited to, mammals including human, birds and the like.

Nasal mucosal administration of the vaccine composition can be performed in an appropriate form. Various methods such as spraying, coating, or direct dripping of a vaccine liquid can be used.

Administration frequency of the vaccine composition of the present invention is at least once, preferably at least twice, in view of the effectiveness. Additional administration is sometimes called booster immunization. Booster immunization makes it possible to achieve a higher infection-protective effect. The interval of booster immunization is recommended to be at least 1 week, preferably 1 to 4 weeks.

The present invention is hereinafter described in more detail by means of the following examples, which, however, are not to be construed as limiting the present invention.

EXAMPLE 1 Nasal Administration Test of Whole Virion Vaccine Containing Ampligen and CVP in Combination for New Type of Influenza, Using Mouse

In the development of an influenza vaccine for nasal administration, a vaccine corresponding to pandemic influenza is highly important and highly urgent as compared to vaccine for seasonal influenza, and needs to be preferentially developed. Therefore, a sample vaccine containing a vaccine for new type of influenza (PR8-IBCDC-RG2 strain: attenuated virus strain of A/Indo/5/2005 (H5N1)) and Ampligen in a 10-fold amount of HA antigen was prepared in the same manner as for seasonal influenza HA vaccine. The sample vaccine contained, as an additive for enhancing an immune response in the nasal mucosa by delaying the clearance of vaccine from the nasal mucosa, a CVP base [mixture of 0.55% CVP, 1.2% L-arginine, 1% glycerin] also applied to allergy medicines.

For a nasal administration test, 7-week-old female BALB/c mice were used (5 per group), and antigen was administered at 0.033-1 μg HA/head. As a comparison control, a group for which Ampligen was added to vaccine in a 10-fold amount of HA antigen and CVP base was not added, a group for which Ampligen was added to vaccine in a 20-fold amount of HA antigen and CVP base was not added, a group for which CVP alone was added to vaccine, a group to which vaccine without addition was nasally administered, and a group to which vaccine without addition was intramuscularly administered were prepared. The vaccine was administered twice at 3-week intervals, and nasal washings and serum were recovered 2 weeks after booster. Specific IgA-ELISA antibody titer of the nasal washings was measured, and the antibody titers of hemagglutination inhibition (HI), neutralizing antibody and specific IgG-ELISA of the serum were measured. Since the sample vaccine had high viscosity, the administration method was changed from conventional nasal drip using a micropipette to injection by inserting about 4 mm of a blunt needle (27G, 0.4 mm×38 mm) set to a microsyringe.

The results are shown in Tables 1-9.

TABLE 1 A/Hiroshima HA vaccine + Poly (I:C) or Ampligen (lot comparison) administra- numbers/ antigen (μg Poly Ampligen group antigen tion route dose group HA or ptn) (I:C) (new) 1 A/Hiroshima/ in  2 μl to each 5 0.1 2 52/2005 nasal cavity 0.1 0.1 3 (H3N2) HA 0.1 1 4 vaccine 2.7 μl to each 0.1 10 nasal cavity 5 0.1 0.1 6 0.1 1 7 0.1 10 8 sc 100 0.1 9 whole virion in 0.2 10 none (control) 10 1st: 1/28 BALB/c mice (7w♀) 2nd: 2/18 Sampling: 2/28 Antigen was A/Hiroshima/52/2005(H3N2) HA vaccine FTHAG0712 (1205 μg HA/mL) or whole virion vaccine (344 μg ptn/mL) poly(I:C)[SIGMA P-0913] was adjusted to 1.88 mg/mL Ampligen (new) was Lot0701HE

TABLE 2 exp. A/Hiroshima HA vaccine + Poly (I:C) or Ampligen NW IgA ELISA group 1 2 3 4 5 6 7 8 9 10 antigen A/Hiroshima/52/2005 (H3N2) HA vaccine whole none virion (control) administration route in sc in dose 2 μl to each nostril 2.7 μl to each nostril 2 μl to each nostril 100 μl 2 μl to each nostril (nasal cavity) (nasal cavity) (nasal cavity) (nasal cavity) numbers/group 5 10 antigen (μg HA or ptn) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 adjuvant Poly (I:C) 0.1 1 10 (μg/head) Ampligen (new) 0.1 1 10 animal 1 1 1 1 16 1 1 128 1 64 1 No. 2 1 32 16 16 128 4 32 1 8 1 3 1 2 16 128 32 32 4 1 8 1 4 1 16 32 256 32 8 16 1 32 1 5 1 64 32 64 2 2 16 1 32 1 geometric mean 1.0 9.2 12.1 55.7 12.1 4.6 21.1 1.0 21.1 1.0 SD 0.0 26.2 13.0 100.6 52.0 12.9 50.6 0.0 23.0 0.0 geometric mean titer (GMT, 2{circumflex over ( )}n) 0.0 3.2 3.6 5.8 3.6 2.2 4.4 0.0 4.4 0.0 positive conversion ratio (≧32, %) 0.0 40.0 40.0 60.0 60.0 20.0 40.0 0.0 60.0 0.0

TABLE 3 exp. A/Hiroshima HA vaccine + Poly (I:C) or Ampligen Serum IgG group 1 2 3 4 5 6 7 8 9 10 antigen A/Hiroshima/52/2005 (H3N2) HA vaccine FTHAG0712 whole none virion (control) administration route in sc in dose 2 μl to each nostril 2.7 μl to each nostril 2 μl to each nostril 100 μl 2 μl to each nostril (nasal cavity) (nasal cavity) (nasal cavity) (nasal cavity) numbers/group 5 10 antigen (μg HA or ptn) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 Poly (I:C) 0.1 1 10 Ampligen (new) 0.1 1 10 animal 1 50 50 100 6400 100 50 1600 51200 1600 50 No. 2 50 800 200 400 400 400 800 51200 400 50 3 50 400 1600 6400 6400 1600 100 51200 200 50 4 50 200 800 3200 200 6400 50 25600 400 100 5 50 400 400 800 200 50 800 25600 1600 50 geometric mean 50.0 263.9 400.0 2111.2 400.0 400.0 348.2 38802.3 606.3 53.6 SD 0.0 282.0 609.9 2906.5 2763.7 2703.5 634.0 14021.7 698.6 15.8 geometric mean titer (GMT, 2{circumflex over ( )}n) 5.6 8.0 8.6 11.0 8.6 8.6 8.4 15.2 9.2 5.7

TABLE 4 exp. A/Hiroshima HA vaccine + Poly (I:C) or Ampligen Serum HI group 1 2 3 4 5 6 7 8 9 10 antigen A/Hiroshima/52/2005 (H3N2) HA vaccine whole none virion (control) administration route in sc in dose 2 μl to each nostril 2.7 μl to each nostril 2 μl to each nostril 100 μl 2 μl to each nostril (nasal cavity) (nasal cavity) (nasal cavity) (nasal cavity) numbers/group 5 10 antigen (μg HA or ptn) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 Poly (I:C) 0.1 1 10 Ampligen (new) 0.1 1 10 animal 1 5 5 5 40 5 5 5 160 5 5 No. 2 5 5 5 5 5 5 5 320 5 5 3 5 5 5 40 5 5 5 320 5 5 4 5 5 5 20 5 10 5 160 5 5 5 5 5 5 5 5 10 5 80 5 5 geometric mean 5.0 5.0 5.0 15.2 5.0 6.6 5.0 183.8 5.0 5.0 SD 0.0 0.0 0.0 17.5 0.0 2.7 0.0 107.3 0.0 0.0 geometric mean titer 2.3 2.3 2.3 3.9 2.3 2.7 2.3 7.5 2.3 2.3 (GMT, 2{circumflex over ( )}n) seroconversion rate (≧40, %) 0.0 0.0 0.0 40.0 0.0 0.0 0.0 100.0 0.0 0.0

TABLE 5 Ampligen pharmacological effect preliminary test: H5 Indonesia whole virion (2008 Sep. 25) test group constitution antigen Ampligen adminis- (μg/ (μg/ CVP tration dose group dose) dose) (%) n route (μl) note 1 0.3 3.00 5 in 2.0 antigen: 2 0.1 1.00 5 in 2.0 Ampligen = 3 0.033 0.33 5 in 2.0 1:10 4 0.3 3.00 0.55 5 in 2.0 antigen: 5 0.1 1.00 0.55 5 in 2.0 Ampligen = 6 0.033 0.33 0.55 5 in 2.0 1:10 + CVP 7 0.3 6.00 5 in 2.0 antigen: 8 0.1 2.00 5 in 2.0 Ampligen = 9 0.033 0.67 5 in 2.0 1:10 10 0.3 0.55 5 in 2.0 antigen + 11 0.1 0.55 5 in 2.0 CVP 12 0.033 0.55 5 in 2.0 13 0.3 5 in 2.0 antigen 14 0.1 5 in 2.0 alone 15 0.033 5 in 2.0 16 0.3 5 im 50.0 antigen alone, intra- muscular control 17 10 in 2.0 saline control 2008 Sep. 30 1st immunization 2008 Oct. 21 2nd immunization 2008 Nov. 4 Sampling Antigen was attenuated virus strain of PR8-IBCDC-RG2 strain: A/Indo/5/2005(H5N1), Lot FPBMW0612-0 3110 μg PTN/mL. about 1048 μg HA/mL)) Ampligen is manufactured by Hemispherx, Lot R-01: 45.7 mg/mL CVP is manufactured by Toko Yakuhin Kogyo, Lot No. INF-9B8, base for preparation (#03-01) Remark 1: prepared for 300 mice antigen: 300 mice × 0.3 μg/head/1.048 μg/μl = 85.9 μl BALB/c mice, 7 w ♀, were used.

TABLE 6 Ampligenpharmacological effect preliminary test: H5 Indonesia whole virion (2008 Sep. 25-) NW IgA (2008 Nov. 6) group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 antigen (μg/dose) 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 Ampligen (μg/dose) 3.00 1.00 0.33 3.00 1.00 0.33 6.00 2.00 0.67 CVP (%) 0.55 0.55 0.55 0.55 0.55 0.55 administration method in in in in in in in in in in in in in in in im in animal 1 32 32 4 256 128 16 8 8 4 16 32 8 16 16 1 1 1 No. 2 16 16 8 512 128 16 32 4 4 32 2 2 4 8 8 1 1 3 32 32 4 128 128 2 16 8 2 16 2 1 16 2 4 1 2 4 16 16 2 64 512 64 32 32 2 64 2 2 8 4 16 2 2 5 8 32 1 64 64 64 64 8 8 4 1 4 8 64 2 2 2 geometric mean 18.4 24.3 3.0 147.0 147.0 18.4 24.3 9.2 3.5 18.4 3.0 2.6 9.2 9.2 4.0 1.3 1.5 titer (GMT) mean titer (2{circumflex over ( )}n) 4.2 4.6 1.6 7.2 7.2 4.2 4.6 3.2 1.8 4.2 1.6 1.4 3.2 3.2 2.0 0.4 0.6 positive conversion 40 60 0 100 100 40 60 20 0 40 20 0 0 20 0 0 0 ratio (titer ≧32, %)

TABLE 7 Ampligen pharmacological effect preliminary test: H5 Indonesia whole virion (2008 Sep. 25-) Serum NT (2008 Nov. 19) group 4 5 6 13 16 antigen (μg/dose) 0.3 0.1 0.033 0.3 0.3 Ampligen (μg/dose) 3.00 1.00 0.33 CVP (%) 0.55 0.55 0.55 Poly L-Arg (μg/dose) administration method in in in in im animal No. 1 80 160 80 20 160 2 1280 10 40 10 80 3 80 160 40 10 320 4 160 160 160 10 80 5 160 160 40 10 640 geometric mean titer (GMT) 183.8 91.9 60.6 11.5 183.8 mean titer (2{circumflex over ( )}n) 7.5 6.5 5.9 3.5 7.5

TABLE 8 Ampligen pharmacological effect preliminary test: H5 Indonesia whole virion (2008 Sep. 25-) Serum HI (2008 Nov.) group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 antigen (μg/dose) 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 Ampligen (μg/dose) 3.00 1.00 0.33 3.00 1.00 0.33 6.00 2.00 0.67 CVP (%) 0.55 0.55 0.55 0.55 0.55 0.55 administration in in in in in in in in in in in in in in in im in method animal No. 1 5 5 5 5 10 5 5 5 5 5 5 5 5 5 5 10 5 2 5 5 5 40 5 5 5 5 5 5 5 5 5 5 5 5 5 3 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 20 5 4 5 5 5 10 10 20 10 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 10 5 5 5 5 5 5 5 5 10 5 geometric mean titer 5.0 5.0 5.0 8.7 6.6 6.6 6.6 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 8.7 5.0 (GMT) mean titer (2{circumflex over ( )}n) 2.3 2.3 2.3 3.1 2.7 2.7 2.7 2.3 2.3 2.3 2.3 2.3 2.3 2.3 2.3 3.1 2.3 seroconversion rate 0 0 0 20 0 0 0 0 0 0 0 0 0 0 0 0 0 (≧40, %)

TABLE 9 Ampligen pharmacological effect preliminary test: H5 Indonesia whole virion (2008 Sep. 25-) Serum IgG (2008 Nov. 6) group 1 2 3 4 5 6 7 8 9 antigen (μg/dose) 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 Ampligen (μg/dose) 3.00 1.00 0.33 3.00 1.00 0.33 6.00 2.00 0.67 CVP (%) 0.55 0.55 0.55 administration in in in in in in in in in method animal No. 1 1600 1600 400 12800 6400 1600 6400 200 50 2 1600 1600 400 51200 6400 800 6400 800 800 3 800 800 200 6400 12800 1600 6400 800 100 4 800 400 200 6400 25600 3200 6400 1600 100 5 800 400 200 12800 12800 3200 12800 800 1600 geometric mean titer 1055.6 800.0 263.9 12800.0 11143.0 1837.9 7351.7 696.4 229.7 (GMT) mean titer (2{circumflex over ( )}n) 10.0 9.6 8.0 13.6 13.4 10.8 12.8 9.4 7.8 positive conversion 100 100 100 100 100 100 100 100 100 ratio (titer ≧32, %) group 10 11 12 13 14 15 16 17 antigen (μg/dose) 0.3 0.1 0.033 0.3 0.1 0.033 0.3 Ampligen (μg/dose) CVP (%) 0.55 0.55 0.55 administration in in in in in in im in method animal No. 1 800 400 400 1600 200 50 12800 50 2 6400 50 50 3200 3200 200 12800 50 3 51200 50 100 1600 50 200 25600 50 4 800 200 100 3200 1600 400 12800 50 5 800 100 200 1600 3200 50 12800 50 geometric mean titer 2785.8 114.9 132.0 2111.2 696.4 132.0 14703.3 50.0 (GMT) mean titer (2{circumflex over ( )}n) 11.4 6.8 7.0 11.0 9.4 7.0 13.8 5.6 positive conversion 100 100 100 100 100 100 100 200 ratio (titer ≧32, %) (Results) Nasal Administration Test of Whole Virion Vaccine for New Type of Influenza, Containing Ampligen and CVP Base in Combination, and Using Mouse

A remarkable immunoenhancing effect was confirmed in the mucosa and serum in the group administered with a vaccine containing Ampligen and a CVP base in combination, which was not found in the group administered with Ampligen alone. The specific IgG, HI and neutralizing antibody titer (NT) of the serum increased to levels equivalent to those of the intramuscular injection group (no adjuvant).

A sample pandemic influenza vaccine containing, as an additive for enhancing an immune response in the nasal mucosa, a CVP base also applied to allergy medicines in combination, was prepared and used for a nasal administration test.

We investigated the convenience of nasal administration method at this time and obtained unexpected effects of a remarkably improved immune response of the nasal mucosa, a specific antibody titer of the serum, which is equivalent to that of intramuscular injection without adjuvant, and the like. Hence, a more practical vaccine was obtained.

EXAMPLE 2 Evaluation System Via Transnasal Immunity in Mouse

(Consideration of Optimal Dose and Administration Frequency for Mouse)

The optimal dose and administration frequency for mouse were examined based on the measurements of serum specific IgG antibody titer and HI antibody titer after addition of Ampligen, CVP and the like to an antigen, and the comparison with existing vaccines having known composition and known administration method, and using a “new type influenza virus antigen at high concentration”.

(Materials)

-   1. New type influenza vaccine stock solution and virus for     evaluation of cross-reactive antibody titer: -   new type influenza vaccine stock solution: lot No.; FPBMW0612-c     (protein concentration: 3110 μg/mL, HA content 992 μg HA/mL, origin     virus strain: A/Indo/5/2005 (H5N1)/PR8-IBCDC-RG2) (The Research     Foundation for Microbial Diseases of Osaka University) -   2. Original strain of virus and inactivated vaccine antigen: for     measurement of specific antibody titer: -   A/Indo/5/2005(H5N1)/PR8-IBCDC-RG2 strain: attenuated strain (clade     2.1) of A/Indonesia/5/2005 (H5N1) for measurement of cross-reactive     antibody titer: -   A/Anhui/01/2005(H5N1)/PR8-IBCDC-RG5 strain: attenuated strain (clade     2.3) of A/Anhui/01/2005 (H5N1) -   A/bar-headed goose/Qinghai/1A/2005 strain: attenuated strain (clade     2.2) of A/Qinghai/1A/2005 (H5N1) -   NIBRG-14 strain: attenuated strain (clade 1) of A/Vietnam/1194/2004     (H5N1) -   3. Adjuvant: -   Ampligen®: containing Ampligen (2.5 mg/mL) manufactured by     Hemispherx -   4. Test method

FIG. 2 shows a schematic view of test flows.

A vaccine containing A/Indo/5/2005 (H5N1)/PR8-IBCDC-RG2 virus antigen (obtained by adding Ampligen to a 20-fold concentration of HA antigen, and adding CVP base (1.1% carboxyvinyl polymer, 2.4% L-arginine, 2.0% glycerol) to 50.0 v/v % of the total amount) was transnasally administered to BALB/c mice and samples (nasal swab and serum) were taken 3 weeks later. In this case, samples were not taken from a part of the mice, and booster was given thereto one to three times. The interval between the boosters for mice subjected to two or three times of boosters was 2 weeks. Samples were taken at 2 weeks from the last booster, and immune response was confirmed when all samples were taken. As comparison controls for the confirmation of the effects of Ampligen and CVP base added, a non-addition vaccine transnasal administration group (high dose, medium dose, low dose) with the same antigen dose but free of Ampligen and CVP base and a negative control group (transnasal administration (once) of saline) were set, and a non-addition vaccine intramuscular administration group (high dose) was set for comparison with vaccines having known dosage form and known administration method. From the above-mentioned test, the relationship between dose and administration frequency, and immune response was evaluated. In addition, immune response with a virus strain having different antigenicity was also investigated to obtain findings as to the cross-reactivity.

-   5. Animals and vaccine (Table 10) -   test animal: BALB/c mouse (♀, 6-week-old when the test was started),     10 mice/group -   trial vaccine for test consideration and adjuvant dose: -   high dose group: [0.3 μg HA+6 μg Ampligen]/dose -   medium dose group: [0.1 μg HA+2 μg Ampligen]/dose -   low dose group: [0.033 μg HA+0.66 μg Ampligen]/dose

As negative control, a saline administration group was set. In addition, for confirmation of effects of vaccine addition, non-addition vaccine transnasal administration group (high dose, medium dose, low dose) with the same antigen dose but free of Ampligen and CVP base was set, and an intramuscular administration group (high dose) was set for the comparison with conventional vaccines (known dosage form•administration method).

-   vaccine composition: -   high dose group: [vaccine 0.3 μL+Ampligen 2.4 μL+CVP base 2.5     μL]/dose -   medium dose group: [vaccine 0.1 μL+Ampligen 0.8 μL+M/75 PBS (PH 7.2)     1.6 μL+CVP base 2.5 μL]]/dose -   low dose group: [vaccine 0.033 μL+Ampligen 0.26 μL+M/75 PBS (PH 7.2)     2.207 μL+CVP base 2.5 μL]/dose -   The vaccine for non-addition transnasal administration used as a     negative control was obtained by removing Ampligen and CVP base from     the above-mentioned composition, and adding M/75 PBS (pH 7.2) in a     volume corresponding thereto. In addition, the vaccine for     non-addition intramuscular administration was [vaccine 0.3 μL+M/75     PBS (pH 7.2) 49.7 μL]/dose. -   Vaccine administration volume: (Table 11)

The transnasal administration group was administered with 6 μL into one nostril, and the control non-addition vaccine subcutaneous administration group was administered with 50 μL to unilateral femoral area.

-   6. Measurement items (Tables 12-19) -   nasal swab: specific IgA-ELISA antibody titer and cross-reactive     IgA-ELISA antibody titer: -   serum specific IgG-ELISA antibody titer, specific HI     (hemagglutination inhibition) antibody titer and cross-reactive HI     antibody titer, specific neutralizing antibody titer and     cross-reactive neutralizing antibody titer were measured. Among the     above-mentioned measurement items, the outline of the measurement     method of the ELISA antibody titer was as described below. -   Vaccine antigen (diluted with 100 mM carbonate buffer (pH 9.6) to     protein concentration of 1 μg/mL) was solid phased (4° C.,     overnight) on a 96-well ELISA plate at 100 μL/well, and washed 3     times with PBS containing 0.1% Tween 20. A sample diluted 2-fold     series or negative control was added at 100 μL/well. Then, the plate     was incubated at 37° C. for 1 hr, and washed 3 times with PBS     containing 0.1% Tween 20. An antibody for detection     (alkaliphosphatase-labeled anti-mouse IgG or biotin-labeled     anti-mouse IgA) was added at 100 μL/well. The plate was incubated at     37° C. for 1 hr, and washed 3 times with PBS containing 0.1% Tween     20 (for IgA detection, alkaliphosphatase-labeled streptavidin was     added at 100 μL/well, incubated at 37° C. for 1 hr, and washed 3     times with PBS containing 0.1% Tween 20). Then,     4-nitrophenylphosphate (1 mg/mL, diluted with 0.1 M diethanolamine     (pH 9.8)) was added at 100 μL/well, and the plate was shaded. After     incubation at room temperature for 30 min, the absorbance at 405 nm     was measured. -   The maximum dilution rate that affords an absorbance of sample     exceeding the average absorbance of negative control+2SD was taken     as the antibody titer of the sample. For specific antibody titer,     the same strain as the administration vaccine antigen was used and,     for cross-reactive antibody titer, vaccine antigen of a different     strain was used. In the following, HI•neutralizing antibody titer     were measured in the same manner. specific HI (hemagglutination     inhibition) antibody titer and cross-reactive HI antibody titer: -   The test was performed according to the description in the “pathogen     detection manual” (edited by National Institute of infectious     Diseases). The red blood cells used were derived from chicken. -   specific neutralizing antibody titer and cross-reactive neutralizing     antibody titer: -   The test was performed according to the description in the “pathogen     detection manual” (edited by National Institute of infectious     Diseases). The culture after virus infection was performed for 4     days. In addition, a method comprising staining the cells with 0.1%     Naphtol Blue Black/0.1% sodium acetate/9% acetic acid, drying the     cell culture plate, adding 0.1 M sodium hydroxide and measuring the     absorbance at 630 nm was used for distinction between virus     infectious cells and non-infectious cells.     (Results)

BALB/c mice (♀, 6.5 w when test was started) were used, and immune response was examined when the antigen (A/Indonesia/5/2005 (H5N1)) supplemented with ampligen at 20-fold ratio of HA antigen, and CVP was transnasally administered 1-4 times, using, as a comparison control, an antigen+Ampligen+CVP transnasal administration group for the first half of the test group and transnasal administration of antigen alone/intramuscular administration of antigen alone/saline transnasal administration for the latter half. The results are shown in Tables 12-19 and FIG. 3.

In the antigen+Ampligen+CVP group, several to dozen-fold mucous membrane IgA/serum IgG and NT antibody titer were observed as compared to the antigen alone transnasal administration group, and the difference was remarkable in the groups with lower dose and less administration frequency.

Conventional common knowledge holds that, as compared to vaccines for subcutaneous or intramuscular administration, transnasal administration-type vaccines show lower specific neutralizing antibody, serum specific IgG antibody titer and HI antibody titer. However, the vaccine evaluated at this time was found to acquire specific neutralizing antibody, serum specific IgG antibody titer and HI antibody titer equivalent to or above those of the vaccines with conventional composition and administration method, by the addition of Ampligen® and the like.

Furthermore, the immune response of the samples obtained by this test with the same strain as the administered antigen and a strain with different antigenicity was examined to confirm cross-reactivity.

TABLE 10 New type Influ A/Indo Ampligen.CVP addition vaccine consideration of dose.administration frequency in mouse; Serum NT test (2009 Aug. 24, Aug. 27) cross-reaction (A/VN) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 antigen 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 amount of administration (μg HA/dose) administration 4 3 2 1 4 frequency administration trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- route nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal adjuvant yes yes yes yes yes yes yes yes yes yes yes yes none none none (Ampligen), CVP addition animal  1 40 20 10 40 160 40 5 40 40 80 80 40 10 40 10 No.  2 20 40 40 80 20 80 10 80 20 80 80 40 40 5 20  3 40 40 10 40 40 160 20 80 20 80 40 20 40 20 20  4 160 40 80 40 10 40 10 40 40 80 40 40 10 80 10  5 80 20 20 40 80 40 40 10 40 80 40 40 10 20 20  6 10 80 10 20 40 20 20 40 40 80 20 10 10 20 20  7 20 10 10 40 40 80 40 40 40 80 10 40 10 20 5  8 40 10 20 80 40 40 10 40 40 80 40 5 20 20 10  9 40 20 40 80 20 80 40 40 80 40 5 20 20 5 10 20 80 80 80 80 40 40 40 20 20 average 34.8 25.9 21.4 45.9 43.2 46.7 18.5 40.0 37.3 80.0 37.3 21.4 17.4 21.4 12.3 antibody titer (GMT) variance 44.2 23.1 27.9 21.6 45.3 44.1 24.0 21.9 16.3 0.0 22.1 16.0 13.7 20.6 6.6 16 17 18 19 20 21 22 23 24 25 26 27 28 29 antigen 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.3 0.3 0.3 none amount of (saline) administration (μg HA/dose) administration 3 2 1 4 3 2 1 1 frequency administration trans- trans- trans- trans- trans- trans- trans- trans- trans- intra- intra- intra- intra- trans- route nasal nasal nasal nasal nasal nasal nasal nasal nasal mus- muscular mus- muscular nasal cular cular adjuvant none none none none none none none none none none none none none none (Ampligen), CVP addition animal  1 40 40 40 20 40 40 40 20 40 80 20 80 80 40 No.  2 40 40 40 40 20 40 20 40 40 20 40 80 40 80  3 40 20 40 20 40 20 40 40 40 80 40 80 40 40  4 20 40 40 40 5 40 40 80 40 80 160 40 5 10  5 80 40 40 40 40 40 20 20 40 20 80 40 40 5  6 80 20 40 20 40 20 20 40 40 40 80 40 40 5  7 40 40 40 20 40 20 40 20 40 40 40 40 40 20  8 20 20 40 20 10 20 40 40 40 160 80 40 10 40  9 20 40 40 40 40 20 20 20 20 20 80 40 80 20 10 80 40 5 40 40 40 40 10 20 80 40 40 20 average 40.0 31.7 40.0 23.0 26.4 28.3 30.3 32.5 32.5 42.9 60.6 40.0 32.5 20.0 antibody titer (GMT) variance 25.0 10.0 0.0 12.5 14.2 10.5 10.3 18.4 10.8 45.0 39.2 19.3 24.3 22.9

TABLE 11 Vaccine composition admini- vaccine stock Ampligen M/75 PBS total dose stration group solution (μL) (μL) CVP (μL) (pH 7.2) (μL) (mL) note high dose 1, 4, 7, 10 302.42 2400.0 3000 297.58 6 for 1000 times (0.3 μg HA/dose) (transnasal) moderate dose 2, 5, 8, 11 100.81 800.0 3000 2099.19 6 for 1000 times (0.1 μg HA/dose) (transnasal) low dose (0.033 μg 3, 6, 9, 12 33.27 264.0 3000 2702.73 6 for 1000 times HA/dose) (transnasal) high dose 13, 16, 19, 22 302.42 5697.58 6 for 1000 times (0.3 μg HA/dose) (transnasal) moderate dose 14, 17, 20, 23 100.81 5899.19 6 for 1000 times (0.1 μg HA/dose) (transnasal) low dose (0.033 μg 15, 18, 21, 24 33.27 5966.73 6 for 1000 times HA/dose) (transnasal) high dose 25, 26, 27, 28 30.24 4969.76 5 for 100 times (0.3 μg HA/dose) (muscular injection) saline 29 600 for 100 times (saline) (transnasal) Whole particles influenza vaccine: A/Indo/5/2005(H5N1) lot No.; FPBMW0612-c (protein concentration: 3110 μg/mL, HA content 992 μg HA/mL) Ampligen (manufactured by Hemispherx): lot; 0701HE (containing 2.5 mg/mL Ampligen) CVP base (manufactured by Toko Yakuhin Kogyo Kabushiki Kaisya): lot INF-N28; carboxyvinyl polymer 1.1%, L-arginine 2.4%, glycerol 2%

TABLE 12 New type Influ A/Indo Ampligen · CVP addition vaccine consideration of mouse dose · administration frequency; Nasal wash IgA-ELISA test (2009 Jun. 19/Jul. 7) group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 antigen 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 amount of administra- tion (μg HA/ dose) ad- 4 3 2 1 4 ministration frequency adminis- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- tration route nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal adjuvant yes yes yes yes yes yes yes yes yes yes yes yes none none none (Ampligen), CVP addition animal  1 512 512 512 256 512 128 1024 512 512 128 1 8 512 512 128 No.  2 512 512 512 256 512 512 4096 512 256 128 64 2 256 1024 64  3 512 1024 256 256 512 512 4096 1024 512 128 4 4 512 128 128  4 512 512 512 2048 512 512 2048 1024 128 128 16 4 512 512 64  5 512 512 512 512 512 512 1024 1024 256 256 64 4 256 128 64  6 512 512 1024 1024 512 128 2048 512 512 32 1 64 512 512 64  7 512 1024 512 128 512 512 2048 512 1024 128 8 4 256 128 64  8 512 512 512 1024 512 512 512 128 256 32 32 2 256 128 64  9 512 512 1024 512 512 2048 128 512 64 1 4 512 128 128 10 512 512 512 512 128 2 4 512 64 64 average 512.0 608.9 512.0 512.0 512.0 376.3 1755.6 474.0 388.0 97.0 6.5 4.9 388.0 222.9 78.8 antibody titer (GMT) variance 0.0 237.0 188.9 588.9 0.0 169.3 1265.7 356.3 250.6 64.4 25.5 19.0 132.2 308.3 30.9 positive 100.0 100.0 100.0 100.0 90.0 90.0 100.0 100.0 100.0 100.0 30.0 10.0 100.0 100.0 100.0 conversion ratio (antibody titer not less than 32, %) group 16 17 18 19 20 21 22 23 24 25 26 27 28 29 antigen amount of 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.3 0.3 0.3 none administration (μg (saline) HA/dose) administration 3 2 1 4 3 2 1 1 frequency administration route trans- trans- trans- trans- trans- trans- trans- trans- trans- intra- intra- intra- intra- trans- nasal nasal nasal nasal nasal nasal nasal nasal nasal muscular muscular muscular muscular nasal adjuvant none none none none none none none none none none none none none none (Ampligen), CVP addition animal No.  1 256 64 64 1024 256 256 64 1 2 8 2 1 1 1  2 128 64 128 256 128 32 32 2 2 2 2 2 1 1  3 128 32 256 512 16 32 32 2 2 2 2 2 1 1  4 512 64 64 1024 128 8 32 2 2 2 2 1 1 1  5 512 64 32 512 128 32 8 1 2 2 2 1 1 1  6 256 128 64 512 64 16 8 8 4 1 2 2 1 1  7 128 64 64 64 16 128 64 4 4 2 4 1 1 1  8 256 32 64 256 16 16 8 4 4 2 2 2 1 1  9 128 64 64 512 32 16 8 8 2 2 2 1 1 1 10 256 32 32 32 16 32 4 4 2 2 1 1 1 average antibody 222.9 59.3 68.6 315.2 52.0 29.9 21.1 2.8 2.6 2.1 2.1 1.3 1.0 1.0 titer (GMT) variance 147.8 27.7 66.1 344.7 78.2 78.6 21.7 2.6 1.0 2.0 0.6 0.5 0.0 0.0 positive conversion 100.0 100.0 100.0 100.0 70.0 50.0 60.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 ratio (antibody titer not less than 32, %)

TABLE 13 New type Influ A/Indo Ampligen.CVP addition vaccine consideration of mouse dose.administration frequency; Serum IgG-ELISA test (2009 Jul. 7) group 1 2 3 4 5 6 7 8 9 antigen 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 amount of administration (μg HA/dose) administration frequency 4 3 2 administration trans- trans- trans- trans- trans- trans- trans- trans- trans- route nasal nasal nasal nasal nasal nasal nasal nasal nasal adjuvant (Ampligen), yes yes yes yes yes yes yes yes yes CVP addition animal No.  1 51200 102400 12800 51200 25600 25600 12800 12800 3200  2 102400 102400 25600 25600 51200 6400 12800 3200 6400  3 102400 12800 25600 51200 25600 1600 12800 1600 3200  4 204800 12800 25600 51200 25600 3200 12800 12800 3200  5 12800 25600 12800 25600 51200 6400 6400 3200 1600  6 25600 51200 51200 51200 25600 6400 6400 12800 3200  7 25600 25600 12800 51200 51200 3200 25600 12800 6400  8 102400 25600 25600 25600 25600 25600 12800 3200 12800  9 102400 102400 51200 51200 25600 6400 3200 6400 10 25600 51200 25600 6400 average 54874.8 33199.1 27437.4 38802.3 34836.2 7465.8 10972.7 5486.4 4525.5 antibody titer (GMT) variance 59259.1 37474.6 27684.1 13219.8 13492.4 10666.7 5938.9 5252.7 3204.4 group 10 11 12 13 14 15 16 17 18 antigen amount 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 of administration (μg HA/dose) administration frequency 1 4 3 administration route trans- trans- trans- trans- trans- trans- trans- trans- trans- nasal nasal nasal nasal nasal nasal nasal nasal nasal adjuvant (Ampligen), CVP yes yes yes none none none none none none addition animal No.  1 800 3200 200 6400 6400 3200 6400 1600 800  2 400 3200 100 6400 6400 1600 3200 3200 3200  3 400 200 400 3200 6400 3200 12800 3200 12800  4 1600 200 200 3200 25600 6400 12800 3200 1600  5 1600 200 400 6400 12800 1600 6400 1600 1600  6 1600 1600 400 6400 25600 1600 6400 800 12800  7 400 200 100 25600 3200 1600 6400 1600 1600  8 6400 6400 100 6400 3200 1600 12800 12800 3200  9 200 200 400 25600 25600 1600 6400 3200 1600 10 400 3200 1600 12800 1600 1600 12800 800 average antibody titer (GMT) 800.0 800.0 263.9 7879.3 7879.3 2111.2 7879.3 2539.8 2425.1 variance 1853.4 2104.1 445.8 8506.6 10064.3 1554.9 3710.4 3622.2 4710.3 group 19 20 21 22 23 24 25 26 27 28 29 antigen 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.3 0.3 0.3 none amount of (saline) administration (μg HA/dose) administration 2 1 4 3 2 1 1 frequency administration trans- trans- trans- trans- trans- trans- intra- intra- intra- intra- trans- route nasal nasal nasal nasal nasal nasal muscular muscular muscular mus- nasal cular adjuvant none none none none none none none none none none none (Ampligen), CVP addition animal  1 1600 1600 1600 200 50 50 25600 51200 3200 1600 50 No.  2 3200 1600 100 200 100 50 12800 51200 6400 400 50  3 3200 200 400 100 100 50 25600 51200 12800 200 50  4 12800 400 100 200 100 50 12800 51200 12800 400 50  5 12800 800 400 200 100 50 12800 204800 6400 400 50  6 1600 400 200 200 100 50 25600 51200 12800 200 50  7 800 100 1600 200 50 50 25600 102400 12800 400 50  8 3200 200 100 100 100 50 12800 102400 12800 400 50  9 3200 200 100 100 100 50 25600 102400 25600 200 50 10 3200 200 100 200 100 50 102400 102400 12800 1600 50 average 3200.0 373.2 246.2 162.5 87.1 50.0 22286.1 77604.7 10396.8 428.7 50.0 antibody titer (GMT) variance 4431.0 577.4 607.5 48.3 21.1 0.0 26849.5 48572.6 6043.4 545.3 0.0

TABLE 14 New type Influ A/Indo Ampligen.CVP addition vaccine consideration of mouse dose.administration frequency; Serum HI test (2009 Jun. 19) group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 antigen 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 amount of administration (μg HA/dose) administration 4 3 2 1 4 frequency administration trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- route nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal adjuvant yes yes yes yes yes yes yes yes yes yes yes yes none none none (Ampligen), CVP addition animal  1 20 5 20 40 20 10 40 20 5 5 10 5 5 5 5 No.  2 5 160 40 20 20 10 5 10 5 5 10 5 5 5 5  3 40 40 10 40 5 5 20 10 5 5 5 10 5 5 5  4 80 10 10 20 10 5 10 40 5 5 5 10 5 5 5  5 20 10 20 40 20 5 5 5 5 10 5 5 5 5 5  6 20 40 5 80 20 5 10 20 5 10 5 5 5 5 5  7 20 5 10 20 20 5 80 20 10 5 5 5 5 5 5  8 40 20 20 5 20 5 40 10 80 20 10 5 5 5 5  9 40 10 20 40 10 40 10 20 5 5 5 10 5 5 10 20 40 10 5 5 10 5 5 5 5 average 24.6 18.3 15.2 23.0 17.1 6.3 18.5 13.6 8.1 6.6 6.6 5.7 5.4 5.0 5.0 antibody titer (GMT) variance 20.9 52.0 12.5 21.7 9.5 2.5 24.8 10.5 23.5 4.9 2.6 2.1 1.6 0.0 0.0 seroconversion 40.0 37.5 20.0 40.0 11.1 0.0 44.4 11.1 10.0 0.0 0.0 0.0 0.0 0.0 0.0 rate (antibody titer not less than 40, %) group 16 17 18 19 20 21 22 23 24 25 26 27 28 29 antigen 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.3 0.3 0.3 none amount (saline) of administration (μg HA/dose) administration 3 2 1 4 3 2 1 1 frequency administration trans- trans- trans- trans- trans- trans- trans- trans- trans- intra- intra- intra- intra- trans- route nasal nasal nasal nasal nasal nasal nasal nasal nasal mus- muscular mus- muscular nasal cular cular adjuvant none none none none none none none none none none none none none none (Ampligen), CVP addition animal No.  1 5 5 5 5 5 10 5 5 5 20 40 5 10 5  2 5 10 5 5 5 5 5 5 5 20 20 5 5 5  3 20 5 5 5 5 5 5 5 5 10 20 20 10 5  4 5 5 5 10 5 5 5 5 5 10 40 10 5 5  5 5 5 5 5 5 5 5 5 5 5 160 5 5 5  6 10 5 5 5 5 5 5 5 5 5 40 5 5 5  7 5 5 5 5 5 5 5 5 5 10 40 10 5 5  8 5 5 5 5 5 5 5 5 5 10 160 10 5 5  9 5 5 5 5 5 5 5 5 5 20 80 20 5 5 10 20 5 5 5 5 5 5 5 10 40 20 10 5 average 7.1 5.4 5.0 5.4 5.0 5.4 5.0 5.0 5.0 10.7 49.2 9.3 6.2 5.0 antibody titer (GMT) variance 6.3 1.7 0.0 1.6 0.0 1.6 0.0 0.0 0.0 5.9 53.2 6.6 2.4 0.0 seroconversion 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 80.0 0.0 0.0 0.0 rate (antibody titer not less than 40, %)

TABLE 15 Serum HI test (2009 Jul. 23) cross-reaction (A/VN) group 4 5 7 8 26 27 antigen amount 0.3 0.1 0.3 0.1 0.3 0.3 of administration (μg HA/dose) administration 3 2 3 2 frequency administration trans- trans- trans- trans- intra- intra- route nasal nasal nasal nasal muscular muscular adjuvant yes yes yes yes none none (Ampligen), CVP addition animal  1 5 20 5 5 5 5 No.  2 5 5 5 5 5 5  3 5 5 5 5 20 5  4 5 5 5 5 10 5  5 5 10 5 5 5 5  6 5 5 5 5 5 5  7 5 20 5 5 5 5  8 5 20 5 5 5 5  9 5 5 5 10 5 5 10 5 5 5 average antibody 5.0 8.6 5.0 5.4 6.2 5.0 titer (GMT) variance 0.0 7.3 0.0 1.7 4.8 0.0 seroconversion 0.0 0.0 0.0 0.0 0.0 0.0 rate (antibody titer not less than 40, %)

TABLE 16 New type Influ A/Indo Ampligen.CVP addition vaccine consideration of mouse dose.administration frequency; Serum NT test (2009 Jul. 27, Aug 3) group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 antigen amount 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 of administration (μg HA/dose) administration 4 3 2 1 4 frequency administration trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- route nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal adjuvant yes yes yes yes yes yes yes yes yes yes yes yes none none none (Ampligen), CVP addition animal  1 1280 5120 640 2560 160 320 640 640 80 80 160 40 320 320 80 No.  2 80 5120 1280 640 640 320 640 160 320 80 320 40 1280 80 40  3 2560 1280 320 2560 320 80 640 320 160 80 40 80 640 80 40  4 2560 320 640 1280 320 80 640 640 80 80 40 80 1280 640 80  5 320 320 320 2560 640 640 320 320 80 80 40 80 1280 160 20  6 640 2560 640 5120 640 80 160 320 160 160 20 10 320 320 20  7 1280 640 320 640 640 80 640 640 160 80 5 40 320 160 20  8 1280 640 2560 160 640 640 320 320 640 320 320 5 80 80 20  9 2560 320 640 1280 320 640 320 160 40 40 5 640 640 10 10 640 2560 320 160 80 160 40 160 10 20 average 905.1 1173.8 685.9 1039.7 508.0 201.6 470.3 373.3 160.0 91.9 60.6 28.3 452.5 149.3 28.3 antibody titer (GMT) variance 947.5 2057.9 892.4 1553.2 320.0 230.2 192.3 180.9 169.7 80.1 121.0 30.0 480.7 229.6 25.5 group 16 17 18 19 20 21 22 23 24 25 26 27 28 29 antigen amount 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.3 0.3 0.3 none of (saline) administration (μg HA/dose) administration 3 2 1 4 3 2 1 1 frequency administration route trans- trans- trans- trans- trans- trans- trans- trans- trans- intra- intra- intra- intra- trans- nasal nasal nasal nasal nasal nasal nasal nasal nasal muscular muscular muscular muscular nasal adjuvant none none none none none none none none none none none none none none (Ampligen), CVP addition animal No.  1 80 80 20 40 80 160 20 10 20 320 640 80 80 20  2 640 160 40 80 80 40 20 20 20 640 320 160 20 20  3 640 20 40 160 40 40 10 20 20 640 320 640 10 20  4 320 80 40 320 40 10 20 20 20 640 640 160 20 10  5 160 40 40 320 40 40 20 20 20 160 640 160 20 5  6 80 40 320 160 40 10 20 20 20 320 640 160 20 5  7 640 20 80 20 20 40 20 20 40 640 640 160 40 10  8 160 320 20 160 20 10 20 20 10 320 2560 160 20 10  9 160 160 40 320 40 20 10 10 10 2560 320 640 40 10 10 640 40 160 40 20 20 20 5 2560 640 1280 20 10 average 259.9 68.6 46.7 130.0 40.0 26.4 17.4 17.4 16.2 597.1 597.1 242.5 24.6 10.7 antibody titer (GMT) variance 256.3 97.7 94.9 113.2 20.7 44.6 4.2 4.2 9.4 902.7 658.4 383.2 20.2 5.9

TABLE 17 New type Influ A/Indo Ampligen.CVP addition vaccine consideration of mouse dose.administration frequency; A/Qinghai strain serum NT test (2009 Nov. 6/9) group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 antigen amount 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 of administration (μg HA/dose) administration 4 3 2 1 4 frequency administration trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- route nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal adjuvant yes yes yes yes yes yes yes yes yes yes yes yes none none none (Ampligen), CVP addition animal  1 160 40 5 80 20 5 40 40 20 20 160 80 160 20 No.  2 40 160 10 320 160 80 20 40 20 20 40 80 20 5 40  3 320 20 10 320 20 40 80 40 20 20 20 80 20 20 40  4 1280 10 20 40 10 10 5 160 40 20 160 40 40 320 20  5 40 5 40 20 40 20 40 20 40 20 40 20 40 80 20  6 20 320 10 160 40 40 80 80 40 40 40 10 40 160 40  7 20 20 10 160 160 5 80 160 40 40 40 10 640 40 5  8 640 10 20 80 20 80 80 80 40 40 160 5 80 80 20  9 40 20 160 80 20 160 80 40 20 20 5 80 80 10 10 80 80 160 40 20 80 20 40 40 40 average 98.5 28.3 16.2 113.1 36.7 25.9 37.0 63.5 34.8 24.6 45.9 23.0 56.6 60.6 21.4 antibody titer (GMT) variance 407.0 112.0 22.5 103.8 59.7 29.4 49.5 51.4 8.4 9.7 54.5 50.1 188.4 93.9 13.4 group 16 17 18 19 20 21 22 23 24 25 26 27 28 29 antigen amount 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.3 0.3 0.3 none of (saline) administration (μg HA/dose) administration 3 2 1 4 3 2 1 1 frequency administration trans- trans- trans- trans- trans- trans- trans- trans- trans- intra- intra- intra- intra- trans- route nasal nasal nasal nasal nasal nasal nasal nasal nasal mus- muscular mus- muscular nasal cular cular adjuvant none none none none none none none none none none none none none none (Ampligen), CVP addition animal No.  1 40 10 10 10 40 40 10 20 10 80 80 40 80 20  2 20 40 20 40 40 20 10 20 10 160 40 80 40 20  3 80 10 5 20 20 10 10 20 20 320 40 80 40 10  4 40 80 20 40 20 20 20 20 20 160 640 40 5 5  5 80 40 40 40 20 40 20 20 20 40 80 80 40 5  6 20 20 5 40 20 20 20 20 20 80 80 160 40 5  7 40 20 20 40 20 20 20 20 20 320 40 80 40 10  8 80 80 20 20 10 5 20 20 40 160 160 40 5 20  9 40 20 10 80 20 10 10 10 10 320 80 160 40 10 10 160 20 80 20 10 20 20 5 40 640 160 20 10 average 49.2 27.2 14.1 34.8 21.4 16.2 15.2 18.7 15.2 130.0 105.6 80.0 26.4 10.0 antibody titer (GMT) variance 42.2 27.4 10.3 23.3 9.5 12.1 5.2 3.2 9.8 114.4 240.8 50.1 21.6 6.3

TABLE 18 New type Influ A/Anhui Ampligen.CVP addition vaccine consideration of mouse dose.administration frequency; Serum NT test (2009 Jul. 27, Aug. 3) group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 antigen amount 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 of administration (μg HA/dose) administration 4 3 2 1 4 frequency administration trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- route nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal adjuvant yes yes yes yes yes yes yes yes yes yes yes yes none none none (Ampligen), CVP addition animal  1 640 160 40 80 320 20 640 160 80 20 80 20 10 20 160 No.  2 80 320 40 320 320 80 80 40 20 40 80 20 80 20 10  3 1280 80 320 320 80 640 80 80 80 40 20 20 20 80 40  4 160 320 80 320 80 40 640 40 40 40 20 40 20 160 20  5 640 80 20 640 320 40 80 80 40 20 20 20 80 20 40  6 320 640 10 640 320 20 80 80 40 40 20 10 80 80 20  7 160 320 80 320 80 40 160 640 20 20 5 20 40 40 10  8 160 10 640 80 320 80 640 160 640 80 20 5 40 10 10  9 640 10 160 40 80 640 40 160 20 10 5 320 40 5 10 20 640 80 160 20 40 20 160 10 20 average 242.5 146.7 69.6 226.3 160.0 58.8 217.7 93.3 69.6 30.3 23.0 15.2 52.8 32.5 20.0 antibody titer (GMT) variance 390.5 203.4 254.9 210.1 132.3 198.2 287.8 190.8 187.2 19.0 27.1 10.1 93.7 47.1 46.1 group 16 17 18 19 20 21 22 23 24 25 26 27 28 29 antigen amount 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.3 0.3 0.3 none of (saline) administration (μg HA/dose) administration 3 2 1 4 3 2 1 1 frequency administration trans- trans- trans- trans- trans- trans- trans- trans- trans- intra- intra- intra- intra- trans- route nasal nasal nasal nasal nasal nasal nasal nasal nasal mus- muscular mus- muscular nasal cular cular adjuvant none none none none none none none none none none none none none none (Ampligen), CVP addition animal No.  1 160 20 40 20 20 40 20 20 20 320 20 80 20 20  2 40 160 40 20 10 20 20 20 20 320 40 80 20 20  3 80 20 40 40 20 80 20 20 20 320 160 40 20 20  4 160 40 40 40 10 20 20 20 20 320 640 160 5 10  5 80 40 40 80 20 20 20 20 20 80 160 80 20 5  6 80 40 20 40 20 10 10 20 20 160 80 40 20 5  7 40 40 40 20 20 10 20 20 20 160 1280 40 20 10  8 160 160 40 40 10 10 20 20 20 320 160 40 5 20  9 20 40 40 80 20 10 20 10 10 160 640 160 20 20 10 80 40 40 20 20 20 20 5 640 640 20 20 20 average 74.6 46.7 37.0 37.3 16.2 18.7 18.7 18.7 16.2 242.5 197.0 60.6 15.2 13.2 antibody titer (GMT) variance 52.7 56.1 6.7 22.0 4.8 21.7 3.2 3.2 5.4 156.6 407.3 49.9 6.3 6.7

TABLE 19 New type Influ A/Indo Ampligen.CVP addition vaccine consideration of mouse dose.administration frequency; Serum NT test (2009 Aug. 24, Aug. 27) cross-reaction (A/VN) group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 antigen amount 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 of administration (μg HA/dose) administration 4 3 2 1 4 frequency administration trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- trans- route nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal nasal adjuvant yes yes yes yes yes yes yes yes yes yes yes yes none none none (Ampligen), CVP addition animal No.  1 40 20 10 40 160 40 5 40 40 80 80 40 10 40 10  2 20 40 40 80 20 80 10 80 20 80 80 40 40 5 20  3 40 40 10 40 40 160 20 80 20 80 40 20 40 20 20  4 160 40 80 40 10 40 10 40 40 80 40 40 10 80 10  5 80 20 20 40 80 40 40 10 40 80 40 40 10 20 20  6 10 80 10 20 40 20 20 40 40 80 20 10 10 20 20  7 20 10 10 40 40 80 40 40 40 80 10 40 10 20 5  8 40 10 20 80 40 40 10 40 40 80 40 5 20 20 10  9 40 20 40 80 20 80 40 40 80 40 5 20 20 5 10 20 80 80 80 80 40 40 40 20 20 average 34.8 25.9 21.4 45.9 43.2 46.7 18.5 40.0 37.3 80.0 37.3 21.4 17.4 21.4 12.3 antibody titer (GMT) variance 44.2 23.1 27.9 21.6 45.3 44.1 24.0 21.9 16.3 0.0 22.1 16.0 13.7 20.6 6.6 group 16 17 18 19 20 21 22 23 24 25 26 27 28 29 antigen amount 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.1 0.033 0.3 0.3 0.3 0.3 none of administration saline) (μg HA/dose) administration 3 2 1 4 3 2 1 1 frequency administration trans- trans- trans- trans- trans- trans- trans- trans- trans- intra- intra- intra- intra- trans- route nasal nasal nasal nasal nasal nasal nasal nasal nasal mus- muscular mus- muscular nasal cular cular adjuvant none none none none none none none none none none none none none none (Ampligen), CVP addition animal No.  1 40 40 40 20 40 40 40 20 40 80 20 80 80 40  2 40 40 40 40 20 40 20 40 40 20 40 80 40 80  3 40 20 40 20 40 20 40 40 40 80 40 80 40 40  4 20 40 40 40 5 40 40 80 40 80 160 40 5 10  5 80 40 40 40 40 40 20 20 40 20 80 40 40 5  6 80 20 40 20 40 20 20 40 40 40 80 40 40 5  7 40 40 40 20 40 20 40 20 40 40 40 40 40 20  8 20 20 40 20 10 20 40 40 40 160 80 40 10 40  9 20 40 40 40 40 20 20 20 20 20 80 40 80 20 10 80 40 5 40 40 40 40 10 20 80 40 40 20 average 40.0 31.7 40.0 23.0 26.4 28.3 30.3 32.5 32.5 42.9 60.6 40.0 32.5 20.0 antibody titer (GMT) variance 25.0 10.0 0.0 12.5 14.2 10.5 10.3 18.4 10.8 45.0 39.2 19.3 24.3 22.9

EXAMPLE 3 Administration of Transnasal Administration-Type Influenza Vaccine (H5N1 Type) and Immune Response Evaluation Test Using Cynomolgus Monkeys (Macaca fascicularis)

Based on the relationship between the administration dose, use and immune response obtained by the experiments using mice in Example 2, administration tests were performed using Cynomolgus monkeys (Macaca fascicularis) as an animal close to human, and the dosage form and composition optimal as a transnasal administration-type vaccine were searched and the usefulness from the aspect of infection defense was investigated.

To be precise, (1) search of optimal dosage form/composition by comparison of immune responses after administration of transnasal administration-type vaccine candidates, and (2) evaluation of practicality from the ability to defend against infection by measurement of serum and mucous membrane antibody titers after vaccine inoculation were performed.

The search was performed by immuno-chemical test of serum (influenza HI antibody titer, IgG-ELISA antibody titer and neutralizing antibody titer) and immuno-chemical test of nasal swab (influenza specific IgA-ELISA antibody titer).

(Test Method)

-   1. Influenza vaccine stock solution: -   Influenza vaccine (H5N1 strain) stock solution (lot No., FPBMQI0813,     protein concentration: 816 μg/mL, HA content 324 μg HA/mL, origin of     virus strain: A/Bar-headed Goose/Qinghai/1A/2005 (H5N1)) -   2. Origin of virus strain and inactivated vaccine antigen to be used     for test -   (for specific antibody titer measurement) -   A/Bar-headed Goose/Qinghai/1A/2005 (H5N1) strain: attenuated strain     (clade 2.2) of A/Qinghai/1A/2005 (H5N1) -   (for cross-reactive antibody titer measurement) -   A/Indo/5/2005 (H5N1)/PR8-IBCDC-RG2 strain: attenuated strain (clade     2.1) of A/Indonesia/5/2005 (H5N1) -   A/Anhui/01/2005 (H5N1)/PR8-IBCDC-RG5 strain: attenuated strain     (clade 2.3) of A/Anhui/01/2005 (H5N1) -   NIBRG-14 strain: attenuated strain (clade 1) of A/Vietnam/1194/2004     (H5N1) -   3. Adjuvant and additive to be used for test -   Ampligen: manufactured by Hemispherx, (containing 10.61 mg/mL of     Ampligen) -   carboxyvinyl polymer (CVP): manufactured by Toko Yakuhin Kogyo     Kabushiki Kaisya, lot; INF-619 (containing 1.1% CVP, 2.4% L-arginine     and 2% glycerol) -   poly L-arginine: manufactured by SIGMA, catalog No. P-3892, lot;     107K5103 (MW>70,000) -   4. Test method

FIG. 4 shows outline of test flows.

TABLE 20 Test group constitution: vaccine Poly HA Ampligen L-Arg antigen (μg/ (μg/ CVP group (μg/dose) dose) dose) addition head note 1 30 — 4 vaccine alone 2 30 600 ∘ 4 trial vaccine 3 30 1500 ∘ 4 Poly L-Arg 4 — — 1 control group

Anesthesized Cynomolgus monkeys (Macaca fascicularis) was transnasally administered with a vaccine containing A/Bar-headed Goose/Qinghai/1A/2005 (H5N1) strain virus antigen or saline at 3 weeks intervals, using a transnasal administration device with trial actuator (test actuator: manufactured by Toko Yakuhin Kogyo Kabushiki Kaisya, Spray Pump; Apta Pharma, VP-7 type, spraying use) six times by 150 μl (50 μl×3) to each nasal cavity (total 300 μl), and samples (nasal swab and serum) were taken at the time of each administration, and every 2 weeks from the booster up to 12 weeks from the booster. Furthermore, where necessary, blood samples nasal swab were taken every 2 weeks thereafter.

Animal and Vaccine:

-   animal: Cynomolgus monkeys (Macaca fascicularis) (male 17 cynomolgus     monkeys, 46-58 months of age when test was started, body weight     2.8-3.5 kg in November 2009), group 1-group 4: 4 cynomolgus     monkeys/group, group 5 (negative control): cynomolgus monkey/group -   trial vaccine for test and adjuvant dose: -   (1) non-addition vaccine group: [30 μg HA]/dose -   (2) Ampligen+CVP addition vaccine group: [30 μg HA+600 μg     Ampligen+0.55% CVP]/dose -   (3) Poly L-arginine+CVP addition vaccine group: [30 μg HA+0.5% Poly     L-arginine+0.55% CVP]/dose -   (4) negative control group: saline -   vaccine administration volume: 150 μl for each nasal cavity, total     300 μl -   5. Measurement items of each sample -   nasal swab: specific IgA-ELISA antibody titer and IgA concentration

The nasal swab was measured for the total IgA concentration and the specific IgA-ELISA antibody titer calculated in the same manner as in Example 2 was amended to be the numerical value per total IgA concentration of 1 μg/mL. serum: specific IgG-ELISA antibody titer, specific, cross-reactive HI (hemagglutination inhibition) antibody titer, specific/cross-reactive neutralizing antibody titer

Among the above-mentioned measurement items, the outline of the measurement method of the ELISA antibody titer was as described below.

Vaccine antigen (diluted with 100 mM carbonate buffer (pH 9.6) to protein concentration of 1 μg/mL) was solid phased (4° C., overnight) on a 96-well ELISA plate at 100 μL/well, and washed 3 times with PBS containing 0.1% Tween 20. A sample diluted 2-fold series or negative control was added at 100 μL/well. Then, the plate was incubated at 37° C. for 1 hr, and washed 3 times with PBS containing 0.1% Tween 20. An antibody for detection (alkaliphosphatase-labeled anti-monkey IgG or biotin-labeled anti-monkey IgA) was added at 100 μL/well. The plate was incubated at 37° C. for 1 hr, and washed 3 times with PBS containing 0.1% Tween 20. The substrate solution (4-NPP or TMB) was added at 100 μL/well and the mixture was shaded and incubated at room temperature for 30 min.

-   A stop solution (2M H₂SO₄ solution for 4-NPP or 650 nm stop solution     for TMB) was added, and the absorbance (405 nm or 655 nm) was     measured.

The maximum dilution rate that affords an absorbance of sample exceeding the average absorbance of negative control+2SD was taken as the antibody titer of the sample. For specific antibody titer, the same strain as the administration vaccine antigen was used and, for cross-reactive antibody titer, vaccine antigen of a different strain was used. In the following, HI antibody titer and neutralizing antibody titer were measured in the same manner.

-   specific HI (hemagglutination inhibition) antibody titer and     cross-reactive HI antibody titer: They were measured in the same     manner as in Example 2. -   specific neutralizing antibody titer and cross-reactive neutralizing     antibody titer: They were measured in the same manner as in Example     2.     (Results)

The results are shown in Tables 21-30 and FIGS. 5-7.

It was confirmed that transnasal spray administration of a vaccine containing Ampligen and CVP to monkey induces specific IgA production in the mucous membrane, and derivation of specific neutralizing antibody in the serum. Since these effects were insufficient with an additive-free vaccine, the usefulness of addition of Ampligen and CVP was confirmed.

TABLE 21 Administration of transnasal administration-type influenza vaccine (H5N1-type) and immune response evaluation test using Cynomolgus monkeys (Macaca fascicularis); Vaccine composition vaccine HA Poly L- CVP M/75 PBS antigen (mL) Ampligen (mL) Arg (mL) addition (pH 7.2) dose group [324 μg HA/mL] [10.61 mg/mL] [100 mg/mL] (mL) (mL) Note (μL) 1 3.087 (30 μg/dose) — — — 6.913 10 mL min 300 2 3.087 30 μg/dose) 1.885 (600 μg/dose) — 5.0 (yes) 0.028 10 mL min 300 3 — — 0.750 (1.5 mg/dose) — 4.250  5 mL min 100 3.704 (30 μg/dose) — — 4 0.296  8 mL min 200 4 — — — 10.000 10 mL min 300 Influenza vaccine stock solution to be used for test influenza vaccine (H5N1 strain) stock solution: lot No.; FPBMQI0813 (protein concentration: 816 μg/mL, HA content 324 μg HA/mL) origin virus strain: A/Bar-headed Goose/Qinghai/1A/2005 (H5N1) adjuvant and additive to be used for test Ampligen (manufactured by Hemispherx): containing 10.61 mg/mL of Ampligen carboxyvinyl polymer (manufactured by Toko Yakuhin Kogyo Kabushiki Kaisya): lot; INF-619 (containing 1.1% CVP (carboxyvinyl polymer), 2.4% L-arginine and 2% glycerol) poly L-arginine (manufactured by SIGMA, catalog No. P-3892): lot; used after dissolving 107K5103 (MW > 70,000) in 100 mg/mL

TABLE 22 Administration of transnasal administration-type influenza vaccine (H5N1-type) and immune response evaluation test (HI) using Cynomolgus monkeys (Macaca fascicularis); before administration (blood samples were collected on 2010 Jan. 28/measured on 2010 Feb. 2) group 1 2 3 4 vaccine HA antigen 30   30   30  — (μg/dose) Ampligen (μg/dose) 600   Poly L-Arg (μg/dose) 1500   CVP addition − + + − animal No. 1 5   5   5  5   2 5   5   5  3 5   5   5  4 5   5   5  average antibody  5.0  5.0   5.0 5.0 titer (GMT) variance  0.0  0.0   0.0 seroconversion rate  0.0  0.0   0.0 0.0 (antibody titer not less than 40, %)

TABLE 23 Administration of transnasal administration-type influenza vaccine (H5N1-type) and immune response evaluation test (HI) using Cynomolgus monkeys (Macaca fascicularis); after once administration (blood samples were collected on 2010 Feb. 18/measured on 2010 Feb. 23) group 1 2 3 4 vaccine HA antigen 30 30 30 — (μg/dose) Ampligen (μg/dose) 600 Poly L-Arg (μg/dose) 1500 CVP addition − + + − animal No. 1 20 5 5 5 2 10 5 5 3 5 5 5 4 5 10 5 average antibody 8.4 5.9 5.0 5.0 titer (GMT) variance 7.1 2.5 0.0 seroconversion rate 0.0 0.0 0.0 0.0 (antibody titer not less than 40, %)

TABLE 24 Administration of transnasal administration-type influenza vaccine (H5N1-type) and immune response evaluation test (HI) using Cynomolgus monkeys (Macaca fascicularis); after twice administration (blood samples were collected on 2010 Mar. 4/measured on 2010 Mar. 9) group 1 2 3 4 vaccine HA antigen 30 30 30 — (μg/dose) Ampligen (μg/dose) 600 Poly L-Arg (μg/dose) 1500 CVP addition − + + − animal No. 1 5 5 5 5 2 5 5 5 3 5 5 5 4 5 5 5 average antibody 5.0 5.0 5.0 5.0 titer (GMT) variance 0.0 0.0 0.0 seroconversion rate 0.0 0.0 0.0 0.0 (antibody titer not less than 40, %)

TABLE 25 Administration of transnasal administration-type influenza vaccine (H5N1-type) and immune response evaluation test (HI) using Cynomolgus monkeys (Macaca fascicularis); 2 weeks after twice administration (blood samples were collected on 2010 Mar. 18/measured on 2010 Mar. 24) group 1 2 3 4 vaccine HA antigen 30 30 30 — (μg/dose) Ampligen (μg/dose) 600 Poly L-Arg (μg/dose) 1500 CVP addition − + + − animal No. 1 5 5 5 5 2 5 5 5 3 5 5 5 4 5 5 5 average antibody 5.0 7.1 5.9 5.0 titer (GMT) variance 0.0 2.9 2.5 seroconversion rate 0.0 0.0 0.0 0.0 (antibody titer not less than 40, %)

TABLE 26 Administration of transnasal administration-type influenza vaccine (H5N1-type) and immune response evaluation test (NT) using Cynomolgus monkeys (Macaca fascicularis); before administration (blood samples were collected on 2010 Jan. 28/measured on 2010 Feb. 2) group 1 2 3 4 vaccine HA antigen 30 30 30 — (μg/dose) Ampligen (μg/dose) 600 Poly L-Arg (μg/dose) 1500 CVP addition − + + − animal No. 1 5 10 5 5 2 5 10 20 3 5 5 10 4 5 5 20 average antibody 5.0 7.1 11.9 5.0 titer (GMT) variance 0.0 2.9 7.5

TABLE 27 Administration of transnasal administration-type influenza vaccine (H5N1-type) and immune response evaluation test (NT) using Cynomolgus monkeys (Macaca fascicularis) after NT administration once (blood samples were 2010 Feb. 18/measured on 2010 Feb. 23) group 1 2 3 4 vaccine HA antigen 30 30 30 — (μg/dose) Ampligen (μg/dose) 600 Poly L-Arg (μg/dose) 1500 CVP addition − + + − animal No. 1 5 10 20 5 2 10 20 5 3 20 20 10 4 5 160 140 average antibody 8.4 28.3 19.3 5.0 titer (GMT) variance 7.1 71.8 64.5

TABLE 28 Administration of transnasal administration-type influenza vaccine (H5N1-type) and immune response evaluation test (NT) using Cynomolgus monkeys (Macaca fascicularis) after NT administration twice (blood samples were collected on 2010 Mar. 4/measured on 2010 Mar. 9) group 1 2 3 4 vaccine HA antigen 30 30 30 — (μg/dose) Ampligen (μg/dose) 600 Poly L-Arg (μg/dose) 1500 CVP addition − + + − animal No. 1 20 20 80 5 2 40 160 5 3 40 160 80 4 10 1280 40 average antibody 23.8 160.0 33.6 5.0 titer (GMT) variance 15.0 587.1 36.1

TABLE 29 Serum IgG ELISA titer after administration of transnasal administration-type influenza vaccine (H5N1-type) using Macaca fascicularis group 1 2 3 4 antibody Pre 141.4 168.2 200.0 100.0 titer Post-3 w 336.4 1131.4 237.8 50.0 Post-5 w 951.4 4525.5 1600.0 200.0 Post-7 w 400.0 5381.7 800.0 100.0

TABLE 30 Nasal swab IgA ELISA titer after administration of transnasaladministration-type influenza vaccine (H5N1-type) using Cynomolgus monkeys (Macaca fascicularis) group 1 2 3 4 antibody Pre 0.7 1.5 1.4 1.1 titer Post-3 w 0.9 1.6 1.3 0.5 Post-5 w 0.7 4.4 2.9 0.6 Post-7 w 2.9 4.3 6.5 0.6

While this invention has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations of the preferred embodiments may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.

All of the references cited herein, including patents, patent applications, and publications, are hereby incorporated in their entireties by reference.

This application is based on a provisional patent application No. 61/165,098 filed in USA (filing date: Mar. 31, 2009), the contents of which are incorporated, in full herein by this reference. 

The invention claimed is:
 1. A composition for nasal mucosal administration, comprising at least one influenza virus antigen, polyriboinosinic polyribocytidylic acid (poly (I:C)) or a derivative thereof and a carboxyvinyl polymer, wherein the weight ratio of the influenza virus antigen and the poly (I:C) or derivative thereof is 1:1-1:50, and wherein the concentration of the carboxyvinyl polymer in the composition is 0.1 to 2.0 wt %.
 2. The composition of claim 1, wherein the influenza virus antigen is a subunit antigen or an inactivated antigen.
 3. The composition of claim 2, wherein the influenza virus antigen comprises at least one subunit antigen selected from the group consisting of HA, NA, M1, M2, NP, PB1, PB2, PA and NS2 of influenza virus.
 4. The composition of claim 2, wherein the influenza virus antigen comprises at least one subunit antigen selected from the group consisting of HA and NA.
 5. The composition of claim 4, wherein the subunit antigen comprises the HA subunit antigen.
 6. The composition of claim 2, wherein the inactivated antigen is a complete virus particle virion.
 7. The composition of claim 1, wherein the concentration of the poly(I:C) in the composition is 0.1 to 10 mg/mL.
 8. The composition of claim 1, wherein the influenza virus antigen is from a subtype selected from the group consisting of H2, H4-H16, and N1-N9.
 9. The composition of claim 1, wherein the influenza virus antigen is of a virus strain selected from the group consisting of A/Hiroshima/52/2005(H3N2), A/Indo/5/2005(H5N1)/PR8-IBCDC-RG2, A/Anhui/01/2005(H5N1)/PR8-IBCDC-RG5, A/Bar-headed Goose/Qinghai/1A/2005 (H5N1) and NIBRG-14.
 10. The composition of claim 1, wherein the influenza antigen comprises an HA antigen which is present at a concentration of 10 to 500 μg/mL in the composition.
 11. The composition of claim 10, wherein the concentration of the HA antigen in the composition is 30 to 400 μg/mL.
 12. The composition of claim 1, which is suitable for administration to mammals or birds.
 13. The composition of claim 12, which is suitable for administration to humans.
 14. The composition of claim 1, wherein the concentration of the carboxyvinyl polymer in the composition is 0.55% and the amount of the poly (I:C) or derivative thereof in the composition is 1 μg/dose.
 15. A method of inducing an immune response against influenza virus in a subject, comprising administering at least once to the nasal mucosa of the subject a composition comprising an effective amount of an influenza virus antigen, poly (I:C) or a derivative thereof, and a carboxyvinyl polymer, wherein the weight ratio of the influenza virus antigen and the poly (I:C) or derivative thereof is 1:1-1:50, and wherein the concentration of the carboxyvinyl polymer in the composition is 0.1 to 2.0 wt %, thereby inducing an immune response against influenza virus in the subject.
 16. The method of claim 15, wherein the influenza virus antigen is a subunit antigen or an inactivated antigen.
 17. The method of claim 16, wherein the influenza virus antigen comprises at least one subunit antigen selected from the group consisting of HA, NA, M1, M2, NP, PB1, PB2, PA and NS2 of influenza virus.
 18. The method of claim 16, wherein the influenza virus antigen comprises at least one subunit antigen selected from the group consisting of HA and NA.
 19. The method of claim 18, wherein the subunit antigen comprises the HA subunit antigen.
 20. The method of claim 16, wherein the inactivated antigen is a complete virus particle virion.
 21. The method of claim 15, wherein the composition is administered at least twice.
 22. The method of claim 21, wherein the composition is administered at an interval of at least 1 week.
 23. The method of claim 15, wherein the antigen comprises an HA antigen which is present at a concentration of 10 to 500 μg/mL in the composition.
 24. The method of claim 23, wherein the concentration of the HA antigen in the composition is 30 to 400 μg/mL.
 25. The method of claim 15, wherein the subject is a mammal or a bird.
 26. The method of claim 25, wherein the subject is a human.
 27. The method of claim 15, wherein the concentration of the carboxyvinyl polymer in the composition is 0.55% and the amount of the poly (I:C) or derivative thereof in the composition is 1 μg/dose. 